However, the ortholog gene of expression system. The average of protein level in each litter of the infected hemolymph on the same immunoblot was normalized to that of C646 time-matched control. Results derived from three self-employed experiments were subjected to standard t test. * 0.05, ** 0.01. (b) Immunoblot analysis of PPOs in hemolymph after baculovirus illness. To compare the amount of cleaved PPOs between infected and control samples when the protein levels of the PPO zymogens in infected samples were comparable to or higher than those in control samples, the amounts of loading samples of Rabbit Polyclonal to PLG 48hI and 72hI were five and seven instances the expected amount respectively in immunoblot assay for antibodies to PPO1, and were four and six instances the expected amount respectively in immunoblot assay for antibodies to PPO2.(TIF) ppat.1006645.s003.tif (364K) GUID:?83D1276A-D618-46FA-9407-35A260FD9A9E S4 Fig: Phylogenetic analysis and RCL region sequences comparison of recognized C646 serpins and serpins of from (Ms) and (Dm). All the selected serpins were aligned and phylogenetic analysis by MEGA 6. Scale pub, 0.1 substitution per site (remaining panel). Alignment of the RCL sequences comprising the scissile relationship and activation sites (right panel). The P1-P1 scissile relationship is definitely indicated by arrow.(TIF) ppat.1006645.s004.tif (186K) GUID:?17BA96B0-7A99-4318-8E46-92B2D4244787 S5 Fig: Phylogenetic analysis of clip-domain serine protease-related proteins (CLIPs). The catalytic website amino acid sequences of 11 (Ha), 3 (Dm), 8 (Bm), 5 (Ms), 2 (Ag), 1 (Tm) and 2 (Tt) CLIPs are compared and divided into three organizations (B~D) based on sequence similarity. Scale pub, 0.1 substitutions per site.(TIF) ppat.1006645.s005.tif (280K) GUID:?544B1646-4FD2-4E34-89D8-0B8E80D9FDF6 S6 Fig: Confirmation of the serpin RNAi silencing efficiency in the protein level and immunoblot analysis of selected proteins. (a) The qPCR analysis. The primers used in qPCR overlapped with the dsRNA related region. * 0.05, ** 0.01. (b) Immunoblot detecting the amounts of VP39, PPO2 cSP4, cSP6, C646 and cSP29. iGFP, GFP dsRNA-treated larvae; iGFP/NPV, GFP dsRNA-treated larvae infected with NPV; iserpin-5/NPV, serpin-5 dsRNA-treated larvae infected with NPV; iserpin-9/NPV, serpin-9 dsRNA-treated larvae infected with NPV. HSP27.2 (warmth shock protein 27.2 kDa) was used as the loading control.(TIF) ppat.1006645.s006.tif (445K) GUID:?5A8A258E-2D13-49B0-BC4E-9CC6D2FD6069 S7 Fig: Ethanol active PPO purified from hemolymph. (a) Immunoblot analysis of 5 ng purified hemolymph PPO using PPO1 and PPO2 antibodies. (b) 2 L hemolymph from na?ve 5th instar larvae and 100 ng purified hemolymph PPO were subjected to native gel and stained by dopamine, which was dissolved in ethanol. (c) 50 ng cSP6Xa was triggered by 400 ng element Xa, and then incubated with 100 ng PPO at space temp for 10 min. the combination was subject to amidase activity assay. Ideals are indicated as mean s.e.m of three indie experiments.(TIF) ppat.1006645.s007.tif (327K) GUID:?56F41B2F-E834-402A-B003-EABCA5B0C3Abdominal S1 Table: Pearson pairwise correlation among biological replicates. (PDF) ppat.1006645.s008.pdf (9.3K) GUID:?5F78E397-03E3-4DAA-8533-AAF0F77E8B87 S2 Table: Identified proteins encoded by baculovirus in infected hemolymph. (PDF) ppat.1006645.s009.pdf (46K) GUID:?2CC09801-DF1F-43FD-AFF1-AF9E84E8E19F S3 Table: A whole list of differential proteins. (PDF) ppat.1006645.s010.pdf (746K) GUID:?C2DF3185-7B2D-45A4-AD32-8850276F15AD S4 Table: Repertoire of baculovirus-regulated immune protein in hemolymph. (PDF) ppat.1006645.s011.pdf (138K) GUID:?95C84D1D-B8C3-4B9A-BAED-7CADDEA7326E S5 Table: List of antibodies produced in this study. (PDF) ppat.1006645.s012.pdf (10K) GUID:?FB2BBB25-FD8E-4E76-8070-3E940BF00201 S6 Table: Primers utilized for qPCR, protein expression, dsRNA synthesis and candida two-hybrid assay. (PDF) ppat.1006645.s013.pdf (18K) GUID:?12B1DF63-DF73-4337-B3E0-FDC2100E8620 Data Availability StatementThe mass spectrometry data (PXD006126) have been deposited in the PRIDE repository (http://www.ebi.ac.uk/pride). All sequence data that support the findings of this study are available in GenBank with the following accession figures: serpin-5 (KY680238), serpin-9 (KY680239), cSP4 (KY680240), cSP6 (KY680241), cSP8 (KY680243), cSP29 (KY680244), PPO1 (KY744277), PPO2 (KY744278). The mass spectrometry data (PXD006126) have been.